Biomarkers for Diagnosis and Prognosis of Corneal Ectatic Disorders

ABSTRACT

The invention relates to the field of the diagnosis and prognosis methods of molecular pathologies. In particular, the invention relates to methods for determining the diagnosis of an ectatic disease of the cornea in a subject, for determining the risk of developing an ectatic disease of the cornea in a subject, for determining the clinical outcome of a subject suffering from an ectatic disease of the cornea and for selecting a subject to be treated with a therapy for an ectatic disease of the cornea based on the determination of the expression levels of TLR2 and/or TLR4 markers. The invention also relates to the use of the TLR2 and/or TLR4 as diagnosis and prognosis markers for an ectatic disease of the cornea.

FIELD OF THE INVENTION

The invention relates to the field of molecular diagnosis and prognosismethods for pathologies. In particular, the invention relates to methodsfor determining the diagnosis of an ectatic disease of the cornea in asubject, for determining the risk of developing an ectatic disease ofthe cornea in a subject, for determining the clinical outcome of asubject suffering from an ectatic disease of the cornea and forselecting a subject to be treated with a therapy for an ectatic diseaseof the cornea based on the determination of the expression levels oftoll-like receptors (TLR) TLR2 and/or TLR4 markers. The invention alsorelates to the use of the TLR2 and/or TLR4 as diagnostic and prognosticmarkers for an ectatic disease of the cornea.

BACKGROUND OF THE INVENTION

Corneal ectasia is a progressive disease that adversely affects thestructural integrity of the cornea. The weakened cornea bulges, andcrippling irregular astigmatism starts to develop. The astigmatismdegrades vision and as the disease progresses, scarring of the corneaoccurs. Corneal ectasia includes keratoconus, pellucid marginaldegeneration, post-refractive surgery ectasia, and other rare diseasessuch as keratoglobus. New modalities for the treatment of cornealectasia have been developed, such as corneal collagen cross-linkage thatuses IN light and Riboflavin to stiffen the cornea and halt theprogression of the disease. It is desirable to halt the progression ofthe disease at a very early stage, before vision is degraded byirregular astigmatism or scarring. The most common cause of thepost-refractive surgery ectasia that threatens vision in those patientsis performing the refractive surgery on an early ectasia patient who wasnot detected by the conventional current diagnostic techniques. Thishighlights the need for a specific and sensitive sign that can be usedto detect those early patients to save them from such a devastatingcomplication.

Corneal topography and thickness are among the current diagnosticcriteria of ectasia. Their use is complicated by their variations amongthe general populations. Normal range of corneal thicknesses is wide,and overlapping between normal thin corneas and early ectasia patientscomplicates the use of this criterion in the diagnosis of early cases ofectasia. Thus, lack of specificity is a significant limitation of usingcorneal thickening for the diagnosis of the ectasia. Corneal topographyuse in diagnosis of ectasia shares the same limitations as cornealthinning. Irregular astigmatism is seen in normal subjects and inectasia patients complicating its use to make the diagnosis, especiallyin mild cases.

Innovative orientation of the research is necessary in this context toprovide biomarkers for diagnosing corneal ectasia in a subject as wellas for determining the risk of onset and the progression of saiddisease.

BRIEF DESCRIPTION OF THE INVENTION

In a first aspect, in vitro method for diagnosing an ectatic disease ofthe cornea in a subject which comprises:

-   -   a) determining the expression level of TLR2 and/or TLR4 in a        sample from said subject; and    -   b) comparing said expression level with a reference value    -   wherein, if the expression level of TLR2 and/or TLR4 is higher        than said reference value is indicative that the subject suffers        from an ectatic disease of the cornea.

In another aspect, the invention relates to an in vitro method fordetermining the risk of developing an ectatic disease of the cornea in asubject which comprises:

-   -   a) determining the expression level of TLR2 and/or TLR4 in a        sample from said subject; and    -   b) comparing said expression level with a reference value.    -   wherein, if the expression level of TLR2 and/or TLR4 is higher        than said reference value is indicative that the subject has        high risk of developing an ectatic disease of the cornea.

In another aspect, the invention relates to an in vitro method fordetermining the clinical outcome of a subject suffering from an ectaticdisease of the cornea, comprising:

-   -   a) determining the expression level of TLR2 and/or TLR4 in a        sample from said subject; and    -   b) comparing said expression level with a reference value    -   wherein if the expression level of TLR2 and/or TLR4 is higher        than said reference value is indicative of a negative clinical        outcome.

In another aspect, the invention relates to an in vitro method forselecting a subject to be treated with a therapy for an ectatic diseaseof the cornea which comprises:

-   -   a) determining the expression level of TLR2 and/or TLR4 in a        sample from said subject; and    -   b) comparing said expression level with a reference value    -   wherein if the expression level of TLR2 and/or TLR4 is higher        than said reference value is indicative that said subject is        candidate to be treated with a therapy for an ectatic disease of        the cornea.

In another aspect the invention relates to the use of TLR2 and/or TLR4as a marker for determining the diagnosis of an ectatic disease of thecornea in a subject.

In another aspect, the invention relates to the use of TLR2 and/or TLR4as a marker for determining the risk of developing an ectatic disease ofthe cornea in a subject.

In another aspect, the invention relates to the use of TLR2 and/or TLR4as a marker for determining the clinical outcome of a subject sufferingfrom an ectatic disease of the cornea.

Finally, in another aspect, the invention relates to the use of TLR2and/or TLR4 as a marker for selecting a subject to be treated with atherapy for an ectatic disease of the cornea.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Median [quartiles] of TLR2 expression levels in corneal cells bystudy groups in cohort A. A gradual increase in the expression of TLR2is observed in a corneal ectasia as keratoconus compared to controlsubjects: Control<Subclinical keratoconus<keratoconus.

FIG. 2: Median [quartiles] of TLR4 expression levels in corneal cells bystudy groups in cohort A. A gradual increase in the expression of TLR2is observed in a corneal ectasia as keratoconus compared to controlsubjects: Control<Subclinical keratoconus<keratoconus.

FIG. 3: Median [quartiles] of TLR2 expression levels in corneal cells bystudy groups in cohort B. A gradual increase in the expression of TLR2is observed: Control<Relatives<keratoconus. Likewise, keratoconus (KC)patients showed a higher expression of TLR2 in corneal cells thanpellucid marginal degeneration (PMD) patients.

FIG. 4: Median [quartiles] of TLR4 expression levels in corneal cells bystudy groups in cohort B. A gradual increase in the expression of TLR4is observed: Control<Relatives<keratoconus. Likewise, keratoconus (KC)patients showed a higher expression of TLR4 in corneal cells thanpellucid marginal degeneration (PMD) patients.

FIG. 5: Median [quartiles] of TLR4 expression levels in conjunctivalcells by study groups in cohort B. A gradual increase in the expressionof TLR4 is observed: Control<Relatives<pellucid marginal degeneration.However, keratoconus (KC) patients showed a similar expression of TLR4in conjunctival cells than pellucid marginal degeneration (PMD)patients.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have discovered that the ectasiaprocess is related with the immunity response. In this regard, theinventors have shown that, unexpectedly, the expression levels of TLR2and TLR4 proteins are increased in subjects diagnosed with an ectasiasuch as keratoconus or pellucid marginal degeneration. Moreover, theinventors of the present patent application have shown a correlationbetween increased expression level of TLR2 and TLR4 and certainparameters which are associated with progression of said disease. Thus,this allows the use of TLR2 and TLR4 not only as diagnostic markers ofthe disease, but also as surrogate markers to predict the risk of onsetand progression of the disease.

Based on these findings, the inventors have developed the methods of thepresent invention in their different embodiments that will be describednow in detail.

Method for Diagnosing Corneal Ectasia in a Subject

In a first aspect, the invention relates to an in vitro method fordiagnosing an ectatic disease of the cornea in a subject, hereinafter,“the first method of the invention”, which comprises:

-   -   a) determining the expression level of TLR2 and/or TLR4 in a        sample from said subject; and    -   b) comparing said expression level with a reference value        wherein, if the expression level of TLR2 and/or TLR4 is higher        than said reference value is indicative that the subject suffers        from an ectatic disease of the cornea.

The term “diagnosis” as used herein, refers both to the process ofattempting to determine and/or identify a possible disease in a subject,i.e. the diagnostic procedure, and to the opinion reached by thisprocess, i.e. the diagnostic opinion. As such, it can also be regardedas an attempt at classification of an individual's condition intoseparate and distinct categories that allow medical decisions abouttreatment and prognosis to be made. As the person skilled in the artwill understand, such a diagnosis may not be correct for 100% of thesubject to diagnose, although preferred it is. The term however requiresthat can identify a statistically significant proportion of subjectsuffering from such pathologies (in this case, corneal ectasia). Theskilled in the art may determine whether a party is statisticallysignificant using different statistical evaluation tools well known, forexample, by determination of confidence intervals, the p-valuedetermination, Student's-test, the Mann-Whitney, etc. Preferredconfidence intervals are at least, 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 95%. The p-values are preferably,0.05, 0.025, 0.001 or lower.

The term “ectatic disease of the cornea” or “corneal ectasia” as usedherein, refers to a condition which involves the progressive thinning ofthe corneal thickness, thus affecting the structural integrity thereofgiving rise to an alteration of the corneal curvature. The weakenedcornea bulges, and crippling irregular astigmatism starts to develop.Corneal ectasia includes keratoconus, (which can be classified insubclinical keratoconus and clinical keratoconus), pellucid marginaldegeneration, keratoglobus and ectasia post-refractive corneal surgery.

The curvature of the cornea can be determined by means of cornealtopography methods such as videokeratography which allows determiningseveral parameters related to corneal curvature such:

-   -   K1: determines the curvature power of the flat meridian of the        anterior surface of the cornea measured within the central 3 mm        zone and expressed in diopters (D);    -   K2: determines the curvature power of the steep meridian of the        anterior surface of the cornea measured within the central 3 mm        zone and expressed in diopters (D);    -   Kc: determines maximum diopter power of the apex.    -   Km: determines the mean curvature power of the anterior surface        of the cornea within the central 3 mm zone expressed in diopters        (D);    -   Kmax: determines the maximum curvature power of the whole        anterior surface of the cornea expressed in diopters (D);    -   Thinnest location: determines the thickness area and the        location of the thinnest point of the cornea (μm).    -   Posterior corneal elevation (μm).

Said parameters measured in healthy subjects can be summarized as: K1(central diopters) between 41.8 and 45.3 D; K2 between 42.2 and 45.3 D;posterior elevation between 0.024-0.036 μm; minimum thickness pointbetween 513.5-567 μm.

In a particular embodiment, the first method of the invention relates toa method for diagnosing an ectatic disease of the cornea, wherein theectatic disease of the cornea is selected from the group consisting ofsubclinical keratoconus, clinical keratoconus, pellucid marginaldegeneration and postrefractive corneal ectasia.

The term “keratoconus” as used herein refers to a progressive disease ofthe cornea causing poor vision, in which the cornea adopts an irregularconical shape due to collagen fiber alteration in its internalstructure. It is bilateral in most cases and progression isasymmetrical. The main anatomical symptom of keratoconus is the thinningin the central or paracentral area. i.e., the cornea becomes thinner askeratoconus develops, which leads to vision deterioration as keratoconusprogresses. Keratoconus can be classified according to thevideokeratographic guidelines proposed by Rabinowitz & McDonnell, it ispossible to classify an eye as “subclinical keratoconus” if thesimulated central corneal power is greater than 47.2 D but less than48.7 D, with an inferior-superior dioptric asymmetry greater than 1.4 D,but less than 1.9 D. Eyes presenting central corneal curvature exceeding48.7 D, as well as inferior-superior dioptric asymmetry greater than 1.9D can be classified as “clinical keratoconus”; 2) The distance visualacuity (DVA) of the subclinical KC eye with value of 1.0 (withoutcorrection or with spherical and/or cylindrical compensation<1.50 D).

The term “pellucid marginal degeneration” or “PMD” as used herein refersto a degenerative corneal condition characterized by a clear bilateralthinning in the inferior and peripheral region of the cornea althoughsome cases affect only one eye. Unlike keratoconus pain is not typicallypresent in pellucid marginal degeneration, and aside from vision loss,no symptoms accompany the condition. However, in rare cases, PMD maypresent with sudden onset vision loss and excruciating eye pain, whichoccurs if the thinning of the cornea leads to perforation. Normally, PMDdoes not present with vascularization of the cornea, scarring, or anydeposits of lipid.

The term “keratoglobus” as used herein refers to a degenerativenon-inflammatory disorder of the eye in which structural changes withinthe cornea cause it to become extremely thin and change to a moreglobular shape than its normal gradual curve. It causes cornealthinning, primarily at the margins, resulting in a spherical, slightlyenlarged eye.

The term “ectasia post refractive corneal surgery” as used herein refersto a condition due to the weakening of the inner layer of the corneawhich occurs during surgery. It results in blurred and distorted visiondue to increased myopia and agtismatism.

The term “TLR2” or “Toll-like receptor 2” as used herein, refers to aprotein that in humans is encoded by the TLR2 gene. TLR2 has also beendesignated as CD282 (cluster of differentiation 282). TLR2 is one of thetoll-like receptors and plays a role in the immune system. Inparticular, the sequence of human TLR2 protein is provided in the NCBIdatabase entry NP 003255 (version of 25 May 2014).

The term “TLR4” or Toll-like receptor 2″ as used herein, refers to aprotein that in humans is encoded by the TLR4 gene. TLR 4 has also beendesignated as CD284 (cluster of differentiation 284). The molecularweight of TLR 4 is approximately 95 kDa. In particular, the sequence ofhuman TLR4 protein is provided in the NCBI database entry NP_003257(version of 25 May 2014).

The term “subject”, as used herein, refers to all animals classified asmammals and includes, but is not restricted to, domestic and farmanimals, primates and humans, e.g., human beings, non-human primates,cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably,the patient is a male or female human of any age or race.

In the present invention, the term “sample” or “biological sample” meansbiological material isolated from a subject's eye. The biological samplecan contain any biological material suitable for detecting the desiredbiomarker and can comprise cell and/or non-cell material of the subject.In a preferred embodiment, the sample is any sample which contains cellsfrom the cornea tissue. In another preferred embodiment, the sample isany sample which contains cells from the conjunctiva tissue. The samplecan be isolated by using any conventional method known in the art.Briefly, corneal epithelial cells may be obtained from a subject bymechanical debridement using a sterile corneal scraper, by impressioncytology or by harvesting cells which are suspended tear samples, suchas corneal epithelial cells released into the tear film by rubbing theeyes and collected with micropipettes using standard techniques, or byany other technique known. Where the sample are conjunctive cells fromthe conjunctive tissue, they can be obtained using know samplingtechniques such as tissue sampled using a micro trephine.

In a preferred embodiment, the first method of the invention comprisesdetermining the expression level of TLR2 in a sample from the subject tobe diagnosed.

In another preferred embodiment, the first method of the inventioncomprises determining the expression level of TLR4 in a sample from thesubject to be diagnosed.

In another preferred embodiment, the first method of the inventioncomprises determining the expression level of TLR2 and TLR4 in a samplefrom the subject to be diagnosed.

As it is used herein, the term “expression level” refers to the value ofa parameter that measures the degree of expression of a specific gene orof the corresponding polypeptide. In a particular embodiment, said valuecan be determined by measuring the mRNA level of the gene of interest ora fragment thereof or by measuring the amount of protein encoded by saidgene of interest or a variant thereof. Thus, in the context of thepresent invention, in a particular embodiment, said expression levelcomprises determining the level of the mRNA encoded from the TLR2 and/orTLR4 gene or determining the level of the TLR2 and/or TLR4 protein.

Virtually any conventional method for detecting and quantifying theexpression level of a gene can be used within the framework of thepresent invention for detecting and quantifying the expression level ofa specific gene. By way of non-limiting illustration, the expressionlevel of a gene can be determined by means of quantifying the mRNA levelof said gene or by means of quantifying the level of protein encoded bysaid gene. Methods for determining the amount of mRNA are well-known inthe state of the art. For example, the nucleic acid contained in thesample, such as the eye's sample from the subject under study, isextracted according to conventional methods, for example, by means ofusing lytic enzymes, chemical solutions or fixing resins. The extractedmRNA can be detected by hybridization (for example by means of Northernblot analysis or DNA or RNA arrays (microarrays) after converting mRNAinto labeled cDNA) and/or amplification by means of a enzymatic chainreaction. In general, quantitative or semi-quantitative enzymaticamplification methods are preferred. The polymerase chain reaction (PCR)or quantitative real-time RT-PCR or semi-quantitative RT-PCR techniqueis particularly advantageous. Primer pairs are preferably designed forthe purpose of superimposing an intron to distinguish cDNA amplificationfrom the contamination from genomic DNA (gDNA). Additional primers orprobes, which are preferably labeled, for example with fluorescence,which hybridize specifically in regions located between two exons, areoptionally designed for the purpose of distinguishing cDNA amplificationfrom the contamination from gDNA. If desired, said primers can bedesigned such that approximately the nucleotides comprised from the 5′end to half the total length of the primer hybridize with one of theexons of interest, and approximately the nucleotides comprised from the3′ end to half the total length of said primer hybridize with the otherexon of interest. Suitable primers can be readily designed by a personskilled in the art. Other amplification methods include ligase chainreaction (LCR), transcription-mediated amplification (TMA), stranddisplacement amplification (SDA) and nucleic acid sequence basedamplification (NASBA). The amount of mRNA is preferably measuredquantitatively or semi-quantitatively. Relevant information aboutconventional methods for quantifying the expression level of a gene canbe found, for example, in Sambrook et al., 2001 [Sambrook, J., et al.,“Molecular cloning: a Laboratory Manual”, 3rd ed., Cold Spring HarborLaboratory Press, N.Y., Vol. 1-3].

To normalize the expression values of one gene among different samples,comparing the mRNA level of the gene of interest (i.e. TLR2 and/or TLR4)in the samples from the subject object of study with a control RNA levelis possible. As it is used herein, a “control RNA” is RNA of a gene theexpression level of which does not differ depending on if the subjectsuffers from an ectatic disease of the cornea or not; a control RNA ispreferably an mRNA derived from a housekeeping gene encoding a proteinthat is constitutively expressed and carrying out essential cellfunctions. Illustrative, non-limiting examples of housekeeping genes foruse in the present invention include GUSB (beta-glucuronidase), PPIA(peptidyl-prolyl isomerase A), β-2-microglobulin, GAPDH, PSMB4(proteasome subunit beta type-4), ubiquitin, transferrin receptor, 18-Sribosomal RNA, cyclophilin, tubulin, β-actin, 3-monooxygenase/tryptophan5-monooxygenase tyrosine activation protein (YWHAZ), etc. If theexpression level of TLR2 and/or TLR4 is determined by measuring theexpression level of transcription product (mRNA) of said gene in asample from the subject under study, the sample can be treated tophysically or mechanically break up the structure of the tissue or cellfor the purpose of releasing the intracellular components into anaqueous or organic solution to prepare the nucleic acids for additionalanalysis. Care is preferably taken to prevent RNA degradation during theextraction process.

In a particular and preferred embodiment of the invention, theexpression level of TLR2 is determined by means of determining theexpression level of the protein encoded by the TLR2 gene or a variantthereof, because increased expression of a gene is usually accompaniedby an increase in the amount of corresponding protein, is also possible.The term “variant” as used herein, relates to those variant of humanTLR2 which appear naturally in other species, i.e. the orthologues ofTLR2. Said variants include, without limitation, mouse TLR2, whichcorresponds to the sequence with accession number NP 036035 dated 25 May2014 in the NCBI database; pig TLR2, which corresponds to the sequencewith accession number NP_998926 dated 10 Jan. 2014 in the NCBI database;macaque TLR2, which corresponds to the sequence with accession numberNP_001123897 dated 2 Mar. 2014 in the NCBI database; rat TLR2, whichcorresponds to the sequence with accession number NP_942064 dated 10Aug. 2014 in the NCBI database.

In another particular and preferred embodiment of the invention, theexpression level of TLR4 is determined by means of determining theexpression level of the protein encoded by the TLR4 gene or a variantthereof. Said variants include, without limitation, mouse TLR4, whichcorresponds to the sequence with accession number NP_067272 dated 4 May2014 in the NCBI database; pig TLR4, which corresponds to the sequencewith accession number NP_001280245 dated 7 Jun. 2014 in the NCBIdatabase; macaque TLR4, which corresponds to the sequence with accessionnumber NP_001032169 dated 26 Feb. 2014 in the NCBI database; rat TLR4,which corresponds to the sequence with accession number NP_062051 dated10 Aug. 2014 in the NCBI database.

The natural variants of TLR2 and/or TLR4 suitable for their use in thepresent invention also derive from said sequence by insertion,substitution or deletion of one or more amino acids and include naturalalleles, variants resulting from alternative processing and truncateforms which appear naturally. The term “variant” also includesfragments, isoforms and analogues or derivatives of TLR2 and/or TLR4.Preferably, variants of TLR2 and/or TLR4 are (i) polypeptides in whichone or more amino acid residues are substituted by a preserved ornon-preserved amino acid residue (preferably a preserved amino acidresidue) and such substituted amino acid may be coded or not by thegenetic code, (ii) polypeptides in which there is one or more modifiedamino acid residues, for example, residues modified by substituentbonding, (iii) polypeptides resulting from alternative processing of asimilar mRNA, (iv) polypeptide fragments and/or (iv) polypeptidesresulting from TLR2 and/or TLR4 fusion or the polypeptide defined in (i)to (iii) with another polypeptide, such as a secretory leader sequenceor a sequence being used for purification (for example, His tag) or fordetection (for example, Sv5 epitope tag). The fragments includepolypeptides generated through proteolytic cut (including multisiteproteolysis) of an original sequence. The variants may bepost-translationally or chemically modified. Such variants are supposedto be apparent to those skilled in the art.

Variants according to the present invention includes amino acidsequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%,or 95% similar or identical to the original amino acid sequence. Thedegree of identity between two proteins is determined using computeralgorithms and methods that are widely known for the persons skilled inthe art. The identity between two amino acid sequences is preferablydetermined by using the BLASTP algorithm [BLASTManual, Altschul, S., etal., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol.Biol. 215: 403-410 (1990)].

The proteins can be post-translationally modified. For example,post-translational modifications that fall within the scope of thepresent invention include signal peptide cleavage, glycosylation,acetylation, isoprenylation, proteolysis myristoylation, protein foldingand proteolytic processing, etc. Additionally, the proteins may includeunnatural amino acids formed by post-translational modification or byintroducing unnatural amino acids during translation.

The determination of the amount of a protein corresponding to theexpression of a specific gene can be performed using any conventionalmethod for protein detection and quantification, for example by means ofan immunoassay, etc. By way of non-limiting illustration, saiddetermination can be performed using antibodies with the capability tobind specifically to the protein to be determined (or fragments thereofwith the antigenic determinants) and subsequent quantification of theantigen-antibody complex derivatives. The antibodies can be, forexample, polyclonal sera, hybridoma supernatants or monoclonalantibodies, fragments of antibodies, Fv, Fab, Fab′ and F(ab′)₂, scFv,diabodies, triabodies, tetrabodies, humanized antibodies, etc. Saidantibodies may (or may not) be labeled with a marker. Illustrative,non-limiting examples of markers that can be used in the presentinvention include radioactive isotopes, enzymes, fluorophores,chemiluminescent reagents, enzyme cofactors, enzyme substrates, enzymeinhibitors, etc. There is a wide range of well-known assays that can beused in the present invention, such as, for example, assays based onWestern-blot or immunoblot techniques, ELISA (enzyme-linkedimmunosorbent assay), RIA (radioimmunoassay), EIA (enzyme immunoassay),DAS-ELISA (double antibody sandwich ELISA), immunocytochemical orimmunohistochemical techniques such as flow cytometry, etc. Other waysof detecting and quantifying the protein include affinitychromatography, ligand binding assay techniques, particle-enhancedturbidimetric immunoassay (PETIA) etc.

In a particular embodiment, the determination of the expression level ofthe TLR2 and/or TLR4 protein is carried out by immunohistochemistry orELISA or protein array. In a preferred embodiment, the expression levelof TLR2 and/or TLR4 is determined by flow cytometry. Briefly, flowcytometry, is a laser-based biophysical technology which allowssimultaneous multiparametric analysis of the physical and chemicalcharacteristics of up to thousands particles per second. Is based inusing fluorescent labels or fluorophores which are typically attached toan antibody that recognizes a target feature on or in the cell; they mayalso be attached to a chemical entity with affinity for the cellmembrane or another cellular structure. Each fluorophore hascharacteristic peak excitation and emission wavelength, and the emissionspectra often overlap. Consequently, the combination of labels which canbe used depends on the wavelength of the lamp(s) or laser(s) used toexcite the fluorochromes and on the detectors available.

If desired, to normalize the expression values of one protein, i.e. TLR2and/or TLR4, among different samples, comparing the protein level of theprotein of interest in the samples from the subject object of study witha control protein level is possible. In one particular embodiment, theexpression level of TLR2 and/or TLR4 is determined in absolute terms,i.e. by providing the concentration of TLR2 and/or TLR4 in a sample. Inanother particular embodiment, the expression level of TLR2 and/or TLR4is measured relative to total protein amount in a sample.

The second step of the first method of the invention comprises comparingthe expression level of TLR2 and/or TLR4 obtained in the first step ofsaid method with a reference value. The term “reference value” as usedherein, refers to a laboratory value used as a reference for thevalues/data obtained from samples obtained from the subjects. Thereference value (or reference level) can be an absolute value, arelative value, a value which has an upper and/or lower limit, a seriesof values, an average value, a median, a mean value, or a valueexpressed by reference to a control or reference value. A referencevalue can be based on the value obtained from an individual sample, suchas, for example, a value obtained from a sample from the subject objectof study but obtained at a previous point in time. The reference valuecan be based on a high number of samples, such as the values obtained ina population of the subjects of the chronological age group coincidingwith that of the subject object of study or based on a set of inclusionor exclusion samples of the sample to be analyzed. The reference valuecan be based on the expression values of the marker to be comparedobtained from samples from healthy subjects who do not have a diseasestate or a particular phenotype. For example, the reference value can bebased on the expression level of the marker to be analyzed obtained fromsubjects who do not have corneal ectasia, preferably from healthysubjects without suffering from any corneal trauma orcorneal-conjunctival disease or any ocular disease. In a preferredembodiment, the reference value is obtained from a sample or a set ofsamples from healthy subjects or subjects without prior history cornealectasia.

The reference value can also be based on the expression values of themarker to be compared obtained from samples from subjects having aparticular phenotype. Once the reference value has been established, theexpression level of TLR2 and/or TLR4 in the sample from the subjectunder study is compared with the reference value. As a consequence ofthis comparison, the expression level of the marker of interest (forexample, TLR2 and/or TLR4 in the first method of the invention) in thesample from the subject can be “greater than”, “less than” or “equal to”said reference value for said gene. In the context of the presentinvention, it is considered that an expression level of TLR2 and/or TLR4in the sample from the subject is “greater than” or “higher than” thereference value for said marker when the expression level of TLR2 and/orTLR4 in the sample from the subject increases, for example, 5%, 10%,25%, 50%, 100% or even more when compared with the reference value forsaid gene, or when it increases, for example, at least 1.1-fold,1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more when comparedwith the reference value for said marker. In the context of the presentinvention, it is also considered that an expression level of the markerof interest (for example, TLR2 and/or TLR4 in the first method of theinvention) in the sample from the subject is “less than” the referencevalue for said marker when the expression level of TLR2 and/or TLR4 inthe sample from the subject decreases, for example, 5%, 10%, 25%, 50%,75%, or even 100% when compared with the reference value for saidmarker.

In the context of the present invention, it is also considered that anexpression level of the marker of interest (i.e. TLR2 and/or TLR4 in thefirst method of the invention) in the sample from the subject is “equalto” the reference value for said marker when the expression level ofTLR2 and/or TLR4 is substantially unchanged with respect to thereference value; for example, it is considered that the expression levelof TLR2 and/or TLR4 in the sample from the subject under study is “equalto” the reference value when the levels differ by not more than 0.1%,not more than 0.2%, not more than 0.3%, not more than 0.4%, not morethan 0.5%, not more than 0.6%, not more than 0.7%, not more than 0.8%,not more than 0.9%, not more than 1%, not more than 2%, not more than3%, not more than 4%, not more than 5%, or not more than the percentagevalue which is the same as the error associated with the experimentalmethod used in the determination.

Once the comparison is made between the expression level of TLR2 and/orTLR4 in the sample from the subject and the reference value for saidmarker, the first method of the invention allows determining if asubject suffers from an ectatic disease of the cornea based on if theexpression level of TLR2 and/or TLR4 is higher than said referencevalue.

In a particular embodiment of the first method of the invention, isaimed at diagnosing a subject as suffering from subclinical keratoconus,in which case the reference value corresponds to the expression levelsof TLR2 and/or TLR4 measured in a sample from a subject from a healthysubject, preferably from subjects who do not have corneal ectasia,preferably from healthy subjects without suffering from any cornealtrauma or corneal-conjunctival disease or any ocular disease. In apreferred embodiment, the reference value is obtained from a sample or aset of samples from healthy subjects or subjects without prior historycorneal ectasia. In this particular embodiment, once the comparison ismade between the expression level of TLR2 and/or TLR4 in the sample fromthe subject and the reference value for said marker, the first method ofthe invention allows determining if a subject suffers from subclinicalkeratoconus based on if the expression level of TLR2 and/or TLR4 ishigher than said reference value. As it is shown in the Examples of thepresent application, values of TLR2 measured by flow cytometry in theconjunctive tissue of healthy subjects are about 213 (90-295) AUF(Arbitrary Fluorescence Units); values of TLR2 measured by flowcytometry in the corneal tissue of healthy subjects are about 185(113-286) AUF; values of TLR4 measured by flow cytometry in theconjunctive tissue of healthy subjects are about 1581 (1281-2265) AUF(Arbitrary Fluorescence Units); values of TLR4 measured by flowcytometry in the corneal tissue of healthy subjects are about 1654(1134-2587) AUF. Therefore, if desired, said values can be used as thereference value in the second step of the first method of the invention.

In another particular embodiment, the first method of the invention isaimed at diagnosing a subject as suffering from clinical keratoconus, inwhich case the reference value corresponds to the expression levels ofTLR2 and/or TLR4 measured in a sample from a subject suffering fromsubclinical keratoconus. In this particular embodiment, once thecomparison is made between the expression level of TLR2 and/or TLR4 inthe sample from the subject and the reference value for said marker, thefirst method of the invention allows determining if a subject suffersfrom clinical keratoconus based on if the expression level of TLR2and/or TLR4 is higher than said reference value. As it is shown in theExamples of the present application, values of TLR2 measured by flowcytometry in the conjunctive tissue of subjects suffering fromsubclinical keratoconus are about 207 (124-398) AUF; values of TLR2measured by flow cytometry in the corneal tissue of subjects sufferingfrom subclinical keratoconus are about 977 (647-1330) AUF; values ofTLR4 measured by flow cytometry in the conjunctive tissue of subjectssuffering from subclinical keratoconus are about 1922 (1574-2778) AUF(Arbitrary Fluorescence Units); values of TLR4 measured by flowcytometry in the corneal tissue of subjects suffering from subclinicalkeratoconus are about 2569 (2071-3808) AUF. Therefore, if desired, saidvalues can be used as the reference value in the second step of thefirst method of the invention.

In yet another particular embodiment, the first method of the inventionis aimed at diagnosing a subject as suffering from pellucid marginaldegeneration, in which case the reference value corresponds to theexpression levels of TLR2 and/or TLR4 measured in a sample from ahealthy subject. In this particular embodiment, once the comparison ismade between the expression level of TLR2 and/or TLR4 in the sample fromthe subject and the reference value for said marker, the first method ofthe invention allows determining if a subject suffers from pellucidmarginal degeneration based on if the expression level of TLR2 and/orTLR4 is higher than said reference value. As it is shown in the Examplesof the present application, values of TLR2 measured by flow cytometry inthe conjunctive tissue of healthy subjects are about 733 (560-946) AUF;values of TLR2 measured by flow cytometry in the corneal tissue ofhealthy subjects are about 580 (413-976) AUF; values of TLR4 measured byflow cytometry in the conjunctive tissue of healthy subjects are about2380 (1781-3283) AUF (Arbitrary Fluorescence Units); values of TLR4measured by flow cytometry in the corneal tissue of healthy subjects areabout 1572 (949-2353) AUF. Therefore, if desired, said values can beused as the reference value in the second step of the first method ofthe invention. In a particular embodiment, the first method of theinvention is aimed at diagnosing a subject as suffering from pellucidmarginal degeneration if the expression level of TLR2 in corneal tissueis higher than said reference value.

In another particular embodiment, the first method of the invention isaimed at diagnosing a subject as suffering from pellucid marginaldegeneration if the expression level of TLR4 in corneal or conjunctivaltissue is higher than said reference value, preferably in conjunctivaltissue.

In another particular embodiment, the first method of the invention alsocomprises determining at least one parameter selected from: diopters,corneal thickness and corneal elevation.

The term “diopter” as used herein refers to a unit of magnifying powerof a lens or lens system. Because the power of a lens is proportional tounity (one) divided by the focal length, the power of a lens in dioptersis numerically equal to 1 m divided by the focal length in meters. Thealgebraic signs of the magnifying power indicate whether the lens causesan incident pencil of parallel light rays to converge or to diverge.Thus, diverging lens having a focal length of 1 m has a power of −1diopter. The diopter can also be used as a measurement of curvatureequal to the reciprocal of the radius measured in meters.

Method for Determining the Risk of Developing an Ectatic Disease of theCornea in a Subject

In a second aspect, the invention relates to an in vitro method fordetermining the risk of developing an ectatic disease of the cornea in asubject, hereinafter, “the second method of the invention” whichcomprises:

-   -   a) determining the expression level of TLR2 and/or TLR4 in a        sample from said subject; and    -   b) comparing said expression level with a reference value        wherein, if the expression level of TLR2 and/or TLR4 is higher        than said reference value is indicative that the subject has        high risk of developing an ectatic disease of the cornea.

The expression “determining the risk” or “prediction of the risk”, orsimilar, as used herein, is synonymous of the expression “assessing therisk” or “assessment of the risk”, means that the present inventionmakes it possible to predict, estimate or evaluate the risk of a subjectto developing an ectatic disease of the cornea. The prediction of riskgenerally implies that the risk is either increased or reduced. As itwill be understood by those skilled in the art, the prediction (or therisk), although preferred to be, need not be correct for 100% of thepatients suffering ectatic diseases of the cornea to be evaluated.

The term, however, requires that a statistically significant portionsubjects can be identified as having an increased probability of havingan ectatic disease of the cornea. Whether a subject is statisticallysignificant can be determined without further ado by the person skilledin the art by using various well known statistic evaluation tools, e.g.,determination of confidence intervals, p-value determination, Student'st-test, Mann-Whitney test, etc. Details can be found in Dowdy andWearden, Statistics for Research, John Wiley & Sons, New York 1983.Preferred confidence intervals are at least 50%, at least 60%, at least70%, at least 80%, at least 90% at least 95%. The p-values are,preferably 0.05, 0.025, 0.001, 0.0001 or lower.

The second method of the invention comprises determining the expressionlevel of TLR2 and/or TLR4 in a sample from said subject.

In a preferred embodiment, the second method of the invention comprisesdetermining the expression level of TLR2 in a sample from the subjectunder study.

In another preferred embodiment, the second method of the inventioncomprises determining the expression level of TLR4 in a sample from thesubject under study.

In another preferred embodiment, the second method of the inventioncomprises determining the expression level of TLR2 and TLR4 in a samplefrom the subject under study.

The terms “subject”, “sample” and “ectatic disease of the cornea” havebeen previously defined in the context of the first method of theinvention and are equally applicable to the second method of theinvention.

In a particular embodiment, the sample from said subject is selectedfrom conjunctival and corneal tissue. Methods for obtaining samples fromconjunctival and corneal tissues have been detailed in the first methodof the invention.

In another particular embodiment, the ectatic disease is selected fromsubclinical keratoconus, clinical keratoconus, pellucid margindegeneration, keratoglobus and ectasia post-refractive corneal surgery.The terms “subclinical keratoconus”, “clinical keratoconus”, “pellucidmargin degeneration”, “keratoglobus” and “ectasia post-refractivecorneal surgery” have been previously defined.

The terms “TLR2”, “TLR4” and “expression level” have been previouslydefined. In a particular embodiment, said expression level comprisesdetermining the level of mRNA encoded from the TLR2 and/or TLR4 gene ordetermining the level of the TLR2 and/or TLR4 protein. Methods fordetermining the expression levels of TLR2 and/or TLR2 have been detailedin the context of the first method of the invention and are equallyapplicable to the second method of the invention.

In a particular embodiment, the determination of the expression level ofthe TLR2 and/or TLR4 protein is carried out by immunohistochemistry orELISA or protein array. In a preferred embodiment, the expression levelof TLR2 and/or TLR4 is determined by flow cytometry.

The second step of the second method of the invention comprisescomparing the expression levels of TLR2 and/or TLR4 with a referencevalue. The term “reference value” has been previously defined in thecontext of the first method of the invention.

Once the comparison is made between the expression level of TLR2 and/orTLR4 in the sample from the subject under study and the reference value,the second method of the invention allows determining if a subject has ahigh risk of developing an ectatic disease of the cornea based on if theexpression level of TLR2 and/or TLR4 is higher than said referencevalue. The terms “higher than”, “less than” and “equal to” have beenpreviously defined.

In a particular embodiment, the second method of the invention is aimedat determining the risk of developing an ectatic disease of the corneain a subject previously diagnosed as having a refractive defect.

The term “refractive defect” as used herein refers to an error in thefocusing of light by the eye on the retina. Refractive defects include:

-   -   myopia which refers to a refractive defect in the eye in which        parallel rays of light from a distance converge at a focal point        located in front of the retina instead of the retina itself, as        it normally would;    -   astigmatism which refers to an eye defect which is characterized        by a different refraction between two meridians of the eye,        which prevents clearly focusing on objects and is generally due        to an impairment in the front curvature of the cornea;    -   hyperopia (or hypermetropia) which is a defect of vision caused        by an imperfection of the eye (often when the eyeball is too        short or the lens cannot become round enough), causing        difficulty focusing on near objects;    -   presbyopia which is a condition associated with aging in which        the eye exhibits a progressively diminished ability to focus        near objects.

In a preferred embodiment, the subject has been previously diagnosed ashaving subclinical keratoconus (simulated central corneal power isgreater than 47.2 D but less than 48.7 D, with an inferior-superiordioptric asymmetry greater than 1.4 D, but less than 1.9 D).

In another particular embodiment of the invention, the subject understudy has developed a previous ocular pathology selected from: ocularitching, eye rubbing, biomicroscopic signs and conjunctival hyperemia.

The term “ocular itching” as used herein refers to ocular pruritus whichmay be caused by atopic keratoconjunctivitis, vernalkeratoconjunctivitis allergic conjunctivitis and atopic dermatitis amongothers. Other causes include dry eye syndrome, meibomian glanddysfunction, blepharithis, contact-lens induced conjunctivitis, giantpapillary conjunctivitis and contact dermatoblepharithis.

The term “biomicroscopic signs” as used herein, refers to abnormalitiesin the areas at the front of the eye, including the eyelids,conjunctiva, iris, lens, sclera, and cornea. The retina and optic nervecan also be seen.

The term “conjunctival hyperemia” as used herein refers to anengorgement of the blood vessels in the conjunctiva due to aninflammation reaction.

Method for Determining the Clinical Outcome of a Subject Suffering froman Ectatic Disease of the Cornea

In a third aspect, the invention relates to an in vitro method fordetermining the clinical outcome of a subject suffering from an ectaticdisease of the cornea, hereinafter, “the third method of the invention”which comprises:

-   -   a) determining the expression level of TLR2 and/or TLR4 in a        sample from said subject; and    -   b) comparing said expression level with a reference value        wherein, if the expression level of TLR2 and/or TLR4 is higher        than said reference value is indicative of a negative clinical        outcome.

The term “determining the outcome” or “predicting the outcome”, is usedherein to refer to the likelihood that a patient will have a particularclinical outcome, whether positive or negative. The predictive methodsof the present invention can be used clinically to make treatmentdecisions by choosing the most appropriate treatment modalities for anyparticular patient. The predictive methods of the present invention arevaluable tools in predicting if a patient is likely to respond favorablyto a treatment regimen, such as refractive surgery. The prediction mayinclude prognostic factors.

As will be understood by those skilled in the art, the prediction,although preferred to be, need not be correct for 100% of the subjectsto be evaluated. The term, however, requires that a statisticallysignificant portion of subjects can be identified as having an increasedprobability of having a given outcome. Whether a subject isstatistically significant can be determined without further ado by theperson skilled in the art using various well known statistic evaluationtools, e.g., determination of confidence intervals, p-valuedetermination, cross-validated classification rates and the like etc.Details are found in Dowdy and Wearden, Statistics for Research, JohnWiley & Sons, New York 1983. Preferred confidence intervals are at least50%, at least 60%, at least 70%, at least 80%, at least 90% or at least95%. The p-values are, preferably, 0.01, 0.005 or lower.

The third method of the invention comprises determining the expressionlevel of TLR2 and/or TLR4 in a sample from said subject.

In a preferred embodiment, the third method of the invention comprisesdetermining the expression level of TLR2 in a sample from the subjectunder study.

In another preferred embodiment, the third method of the inventioncomprises determining the expression level of TLR4 in a sample from thesubject under study.

In another preferred embodiment, the third method of the inventioncomprises determining the expression level of TLR2 and TLR4 in a samplefrom the subject under study.

The first step of the third method of the invention comprisesdetermining the expression levels of TLR2 and/or TLR4 in a sample forthe subject under study.

The terms “subject”, “sample” and “ectatic disease of the cornea” havebeen previously defined in the context of the first method of theinvention and are equally applicable to the third method of theinvention.

In a particular embodiment, the sample from said subject is selectedfrom conjunctival and corneal tissue. Methods for obtaining samples fromconjunctival and corneal tissues have been detailed in the first methodof the invention.

In another particular embodiment, the ectatic disease is selected fromsubclinical keratoconus, clinical keratoconus, pellucid margindegeneration, keratoglobus and ectasia post refractive corneal surgery.The terms “subclinical keratoconus”, “clinical keratoconus”, “pellucidmargin degeneration”, “keratoglobus” and “ectasia post refractivecorneal surgery” have been previously defined.

The terms “TLR2”, “TLR4” and “expression level” have been previouslydefined. In a particular embodiment, said expression level comprisesdetermining the level of mRNA encoded from the TLR2 and/or TLR4 gene ordetermining the level of the TLR2 and/or TLR4 protein. Methods fordetermining the expression levels of TLR2 and/or TLR2 have been detailedin the context of the first method of the invention and are equallyapplicable to the third method of the invention.

In a particular embodiment, the determination of the expression level ofthe TLR2 and/or TLR4 protein is carried out by immunohistochemistry orELISA or protein array. In a preferred embodiment, the expression levelof TLR2 and/or TLR4 is determined by flow cytometry.

The second step of the third method of the invention comprises comparingthe expression levels of TLR2 and/or TLR4 with a reference value. Theterm “reference value” has been previously defined. In a particularembodiment, said reference value is obtained from a sample or a set ofsamples from healthy subjects or subjects without prior history cornealectasia.

In another particular embodiment, said reference value refers to theTLR2 and/or TLR4 expression levels in a sample from said subjectobtained at earlier point of time. Thus, according to the third methodof the invention, the expression levels of TLR2 and/or TLR4 determinedin a sample from a subject suffering from an ectatic disease of thecornea obtained at first time are compared with the expression levels ofTLR2 and/or TLR4 determined in a sample from a subject suffering from anectatic disease of the cornea obtained at a second period of time. Thesecond subjects sample can be taken at any time after the first periodof time, e.g., one day, one week, one month, two months, three months,six months 1 year, 2 years, 5 years, 10 years or more after the firstsubject sample.

Lastly, the subject is then classified as having a negative outcome ifthe expression level of TLR2 and/or TLR4 is higher than said expressionlevels in the reference sample.

The term “positive outcome” in relation to ectatic disease of the corneameans that the degeneration of the cornea does not advance. Said termalso encompass a decrease in the cornea degeneration rate.

The term “negative outcome” in relation to ectatic disease of the corneameans that the degeneration of the cornea progresses.

Method for Selecting a Subject to be Treated with a Therapy for anEctatic Disease of the Cornea

In a fourth aspect, the invention relates to an in vitro method forselecting a subject to be treated with a therapy for an ectatic diseaseof the cornea, hereinafter, “the fourth method of the invention” whichcomprises:

-   -   a) determining the expression level of TLR2 and/or TLR4 in a        sample from said subject; and    -   b) comparing said expression level with a reference value        wherein, if the expression level of TLR2 and/or TLR4 is higher        than said reference value is indicative that said subject is        candidate to be treated with a therapy for ab ectatic disease of        the cornea.

The term “selecting a subject for a therapy”, as used herein, relates tothe identification of a subject for a therapy designed to cure a diseaseor palliate the symptoms associated with one or more diseases orconditions. In the particular case of ectatic disease of the cornealtherapy, it is understood any therapy which abolishes, retards orreduces the symptoms associated with corneal ectasia. Adequate therapieswhich can be used according to the invention to treat ectatic diseasesof the cornea include corneal crosslinking, intracorneal rings andrefractive surgery.

Corneal crosslinking is a technique which uses ultraviolet light orlight in the blue spectrum and a photosensiziter to strengthen chemicalbonds in the cornea and thereby increase the corneal stiffness.

Intrastromal corneal rings or intracorneal rings are small devicesimplanted in the eye to correct vision. A typical vision correctionusing corneal rings would involve an ophthalmologist making a smallincision in the cornea of the eye and inserting two crescent orsemi-circular shaped rings segments between the layers of the cornealstroma, one on each side of the pupil. The embedding of the rings in thecornea has the effect of flattening the cornea and changing therefraction of light passing through the cornea on its way into the eye.

The refractive eye surgery is used to improve the refractive state ofthe eye and decrease or eliminate dependency on glasses or contactlenses. This can include various methods of surgical remodeling of thecornea. The most common methods today, such as Automated LamellarKeratoplasty (ALK), Laser-assisted in situ Keratomileusis (LASIK) orReLEx, use excimer lasers to reshape the curvature of the cornea.

The fourth method of the invention comprises determining the expressionlevel of TLR2 and/or TLR4 in a sample from said subject.

In a preferred embodiment, the fourth method of the invention comprisesdetermining the expression level of TLR2 in a sample from the subjectunder study.

In another preferred embodiment, the fourth method of the inventioncomprises determining the expression level of TLR4 in a sample from thesubject under study.

In another preferred embodiment, the fourth method of the inventioncomprises determining the expression level of TLR2 and TLR4 in a samplefrom the subject under study.

The first step of the fourth method of the invention comprisesdetermining the expression levels of TLR2 and/or TLR4 in a sample forthe subject under study.

The terms “subject”, “sample” and “ectatic disease of the cornea” havebeen previously defined in the context of the first method of theinvention and are equally applicable to the fourth method of theinvention.

In a particular embodiment, the sample from said subject is selectedfrom conjunctival and corneal tissue. Methods for obtaining samples fromconjunctival and corneal tissues have been detailed in the first methodof the invention.

In another particular embodiment, the ectatic disease is selected fromsubclinical keratoconus, clinical keratoconus, pellucid margindegeneration, keratoglobus and ectasia post refractive corneal surgery.The terms “subclinical keratoconus”, “clinical keratoconus”, “pellucidmargin degeneration”, “keratoglobus” and “ectasia post refractivecorneal surgery” have been previously defined.

The terms “TLR2”, “TLR4” and “expression level” have been previouslydefined. In a particular embodiment, said expression level comprisesdetermining the level of mRNA encoded from the TLR2 and/or TLR4 gene ordetermining the level of the TLR2 and/or TLR4 protein. Methods fordetermining the expression levels of TLR2 and/or TLR2 have been detailedin the context of the first method of the invention and are equallyapplicable to the third method of the invention.

In a particular embodiment, the determination of the expression level ofthe TLR2 and/or TLR4 protein is carried out by immunohistochemistry orELISA or protein array. In a preferred embodiment, the expression levelof TLR2 and/or TLR4 is determined by flow cytometry.

The second step of the fourth method of the invention comprisescomparing the expression levels of TLR2 and/or TLR4 with a referencevalue. The term “reference value” has been previously defined. In aparticular embodiment, said reference value is obtained from a sample ora set of samples from healthy subjects or subjects without prior historycorneal ectasia.

Once the comparison is made between the expression level of TLR2 and/orTLR4 in the sample from the subject under study and the reference value,the fourth method of the invention allows selecting a subject to betreated with a therapy for an ectatic disease of the cornea based on ifthe expression level of TLR2 and/or TLR4 is higher than said referencevalue. The terms “higher than”, “less than” and “equal to” have beenpreviously defined.

Method for Selecting a Refractive Surgery as Therapy for a SubjectSuffering from a Refractive Defect

In a fifth aspect, the invention relates to an in vitro method fordetermining whether a subject suffering from a refractive defect is acandidate for a refractive surgery therapy which comprises:

a) determining the expression level of TLR2 and/or TLR4 in a sample fromsaid subject; and

b) comparing said expression level with a reference value wherein if theexpression level of TLR2 and/or TLR4 is higher than said reference valueis indicative that said subject is not candidate to be treated with arefractive surgery for said refractive defect.

In a preferred embodiment, the fifth method of the invention comprisesdetermining the expression level of TLR2 and TLR4 in a sample from thesubject under study.

The term “refractive surgery” as used herein refers to any eye surgeryused to improve the refractive state of the eye based on remodeling thecurvature of the cornea.

The first step of the fifth method of the invention comprisesdetermining the expression levels of TLR2 and/or TLR4 in a sample forthe subject under study. The terms “subject”, “sample” and “refractivedefect” have been previously defined in the context of the first methodof the invention and are equally applicable to the fifth method of theinvention.

In a particular embodiment, the sample from said subject is selectedfrom conjunctival and corneal tissue. Methods for obtaining samples fromconjunctival and corneal tissues have been detailed in the first methodof the invention.

In a particular embodiment, the determination of the expression level ofthe TLR2 and/or TLR4 protein is carried out by immunohistochemistry orELISA or protein array. In a preferred embodiment, the expression levelof TLR2 and/or TLR4 is determined by flow cytometry.

The second step of the fifth method of the invention comprises comparingthe expression levels of TLR2 and/or TLR4 with a reference value. Theterm “reference value” has been previously defined.

Once the comparison is made between the expression level of TLR2 and/orTLR4 in the sample from the subject under study and the reference value,the fourth method of the invention allows selecting a subject to betreated with a therapy for an ectatic disease of the cornea based on ifthe expression level of TLR2 and/or TLR4 is higher than said referencevalue. The terms “higher than”, “less than” and “equal to” have beenpreviously defined.

Uses of the Invention

In a sixth aspect, the invention relates to the use of TLR2 and/or TLR4as a marker for determining the diagnosis of an ectatic disease of thecornea.

In a seventh aspect, the invention relates to the use of TLR2 and/orTLR4 as a marker for determining the risk of developing an ectaticdisease of the cornea in a subject.

In an eight aspect, the invention relates to the use of TLR2 and/or TLR4as a marker for determining the clinical outcome of a subject sufferingfrom an ectatic disease of the cornea.

Finally, the invention relates the invention relates to the use of TLR2and/or TLR4 as a marker for selecting a subject to be treated with atherapy for an ectatic disease of the cornea or for selecting arefractive surgery as therapy for a subject suffering from a refractivedefect.

The terms “TLR2”, “TLR4”, “diagnosis”, “determining the risk”,“determining the clinical outcome”, “selecting a subject to be treatedwith therapy”, “ectatic disease of the cornea” and their particulars andpreferred embodiments have been previously defined in defined in thecontext of the first, second, third and fourth method of the inventionand are equally applicable to the uses of the invention.

Method for Determining the Risk that a Patient Suffers Ectasia FollowingRefractive Surgery

In another aspect, the invention relates to an in vitro method fordetermining the risk that a patient suffers ectasia following refractivesurgery which comprises:

-   -   a) determining the expression level of TLR2 and/or TLR4 in a        sample from said subject; and    -   b) comparing said expression level with a reference value        wherein if the expression level of TLR2 and/or TLR4 is higher        than said reference value is indicative that said subject shows        high risk of suffering ectasia following refractive surgery.

In a preferred embodiment, the method of the invention comprisesdetermining the expression level of TLR2 and TLR4 in a sample from thesubject under study.

The term “refractive surgery” as used herein refers to any eye surgeryused to improve the refractive state of the eye based on remodeling thecurvature of the cornea.

The first step of the fifth method of the invention comprisesdetermining the expression levels of TLR2 and/or TLR4 in a sample forthe subject under study.

The terms “subject”, “sample” and “refractive defect” have beenpreviously defined in the context of the first method of the inventionand are equally applicable to the fifth method of the invention.

In a particular embodiment, the sample from said subject is selectedfrom conjunctival and corneal tissue. Methods for obtaining samples fromconjunctival and corneal tissues have been detailed in the first methodof the invention.

In a particular embodiment, the determination of the expression level ofthe TLR2 and/or TLR4 protein is carried out by immunohistochemistry orELISA or protein array. In a preferred embodiment, the expression levelof TLR2 and/or TLR4 is determined by flow cytometry.

The second step of the fifth method of the invention comprises comparingthe expression levels of TLR2 and/or TLR4 with a reference value. Theterm “reference value” has been previously defined.

Once the comparison is made between the expression level of TLR2 and/orTLR4 in the sample from the subject under study and the reference value,the fourth method of the invention allows selecting a subject to betreated with a therapy for an ectatic disease of the cornea based on ifthe expression level of TLR2 and/or TLR4 is higher than said referencevalue. The terms “higher than”, “less than” and “equal to” have beenpreviously defined.

The Present Invention is Also Directed to:

-   -   1. An in vitro method for diagnosing an ectatic disease of the        cornea in a subject which comprises:        -   a) determining the expression level of TLR2 and/or TLR4 in a            sample from said subject; and        -   b) comparing said expression level with a reference value            wherein, if the expression level of TLR2 and/or TLR4 is            higher than said reference value is indicative that the            subject suffers from an ectatic disease of the cornea.    -   2. The in vitro method according to aspect 1 wherein said        ectatic disease of the cornea is selected from subclinical        keratoconus, clinical keratoconus, pellucid marginal        degeneration, keratoglobus and ectasia post refractive corneal        surgery.    -   3. The in vitro method according to aspect 2, wherein said        ectatic disease of the cornea is subclinical keratoconus, in        which case the reference value corresponds to the expression        levels of TLR2 and/or TLR4 measured in a sample from a healthy        subject.    -   4. The in vitro method according to aspect 2, wherein said        ectatic disease of the cornea is clinical keratoconus, in which        case the reference value corresponds to the expression levels of        TLR2 and/or TLR4 measured in a sample from a subject suffering        from subclinical keratoconus.    -   5. The in vitro method according to aspect 2, wherein said        ectatic disease of the cornea is pellucid marginal degeneration,        in which case the reference value corresponds to the expression        levels of TLR2 and/or TLR4 measured in a sample from a healthy        subject.    -   6. The in vitro method according to aspects 1 to 5, wherein the        sample from said subject is selected from conjunctival and        corneal tissue.    -   7. The in vitro method according to aspects 1 to 6, wherein said        expression level comprises determining the level of mRNA encoded        from the TLR2 and/or TLR4 gene or determining the level of the        TLR2 and/or TLR4 protein.    -   8. The in vitro method according to aspect 7, the protein level        is determining by immunohistochemistry, Western blot, flow        cytometry or by ELISA.    -   9. The in vitro method of aspects 1 to 8 which also comprises        determining at least one parameter selected from: diopters,        corneal thickness and corneal elevation.    -   10. An in vitro method for determining the risk of developing an        ectatic disease of the cornea in a subject which comprises:        -   a) determining the expression level of TLR2 and/or TLR4 in a            sample from said subject; and        -   b) comparing said expression level with a reference value.        -   wherein, if the expression level of TLR2 and/or TLR4 is            higher than said reference value is indicative that the            subject has high risk of developing an ectatic disease of            the cornea.    -   11. The in vitro method according to aspect 10, wherein the        subject has been previously diagnosed as having a refractive        defect.    -   12. The in vitro method according to aspect 10 or 11, wherein        said subject has developed a previous ocular pathology selected        from: ocular itching, eye rubbing, biomicroscopic signs and        conjunctival hyperemia.    -   13. An in vitro method for determining the clinical outcome of a        subject suffering from an ectatic disease of the cornea,        comprising:        -   a) determining the expression level of TLR2 and/or TLR4 in a            sample from said subject; and        -   b) comparing said expression level with a reference value        -   wherein if the expression level of TLR2 and/or TLR4 is            higher than said reference value is indicative of a negative            clinical outcome.    -   14. An in vitro method for selecting a subject to be treated        with a therapy for an ectatic disease of the cornea which        comprises:        -   a) determining the expression level of TLR2 and/or TLR4 in a            sample from said subject; and        -   b) comparing said expression level with a reference value        -   wherein if the expression level of TLR2 and/or TLR4 is            higher than said reference value is indicative that said            subject is candidate to be treated with a therapy for an            ectatic disease of the cornea.    -   15. An in vitro method according to aspect 14 wherein said        therapy is selected from corneal crosslinking, intracorneal        rings and refractive surgery.    -   16. An in vitro method for selecting a refractive surgery as        therapy for a subject suffering from a refractive defect which        comprises:        -   a) determining the expression level of TLR2 and/or TLR4 in a            sample from said subject; and        -   b) comparing said expression level with a reference value        -   wherein if the expression level of TLR2 and/or TLR4 is            higher than said reference value is indicative that said            subject is not candidate to be treated with a refractive            surgery for said refractive defect.    -   17. The in vitro method according to any of aspects 10 to 16,        wherein said ectatic disease of the cornea is selected from        subclinical keratoconus, clinical keratoconus, pellucid margin        degeneration, keratoglobus and ectasia post refractive corneal        surgery.    -   18. The in vitro method according to aspects 10 to 17, wherein        the sample from said subject is selected from conjunctival and        corneal tissue.    -   19. The in vitro method according to aspects 10 to 18, wherein        said expression level comprises determining the level of mRNA        encoded from the TLR2 and/or TLR4 gene or determining the level        of the TLR2 and/or TLR4 protein.    -   20. The in vitro method according to aspect 19, the protein        level is determining by immunohistochemistry, Western Blot, flow        cytometry or ELISA.    -   21. Use of TLR2 and/or TLR4 as a marker for determining the        diagnosis of an ectatic disease of the cornea in a subject, for        determining the risk of developing an ectatic disease of the        cornea in a subject, as a marker for determining the clinical        outcome of a subject suffering from an ectatic disease of the        cornea or as a marker for selecting a subject to be treated with        a therapy for an ectatic disease of the cornea.

The following example is provided as merely illustrative and is not tobe construed as limiting the scope of the invention.

EXAMPLES Materials and Methods

We have included 2 different cohorts of patients:

-   -   1) In a cohort A we have included unilateral KC patients and        control subjects in order to test whether TLR2 and TLR4        expression in cells of the corneal epithelium and bulbar        conjunctiva of patients with pathological or clinical KC is        greater than in subclinical KC, and higher than in samples from        control subjects.    -   2) In a cohort B we have included bilateral KC patients,        relatives of patients, PMD patients and control subjects in        order to test whether TLR2 and TLR4 expression in cells of the        corneal epithelium and bulbar conjunctiva of patients with        pathological or clinical KC is greater than in their relatives,        and higher than in samples from control subjects. Likewise, we        tested whether TLR2 and TLR4 expression in cells of the corneal        epithelium and bulbar conjunctiva of PMD patients is greater        than in control subjects.

Patients and Control Subjects in Cohort A

We have designed a prospective, cross sectional study in which 50unilateral KC patients (50 KC and 50 subclinical KC eyes) and 19 controlsubjects (38 eyes) were enrolled. Unilateral KC patients (64% males;mean age, 33.3±9.5 years) were selected from a database of KC patientsat Servizo Galego de Saúde, Complexo Hospitalario de Santiago deCompostela, Spain. We studied both eyes from each patient or controlsubject. Patients were asked not to wear their contact lenses for a weekprior to the study. Control (45.9% males; mean age, 28.5±4.7 years)subjects had not previous history of KC or eye rubbing, and notopographic alterations. All patients and control subjects wereexpressly cited for the purposes of this study, and all examinationswere performed by the same researcher. Data collected included gender,age, patient's ocular history, medical history (allergy, eye rubbing),and history of KC. This research was carried out in accordance with theDeclaration of Helsinki of the World Medical Association (2008) andapproved by the Ethics Committee of Research at Servizo Galego de Saúde.Informed consent was obtained from each patient or control subject afterfull explanation of the procedures.

Inclusion criteria were: 1) Asymmetric KC patients (1 KC eye and 1subclinical KC eye). Rabinowitz/McDonnell criterion was used for the KCdiagnosis (Rabinowitz Y. S. Keratoconus. Sury Ophthalmol. 1998;42:297-319). According to the videokeratographic guidelines proposed byRabinowitz & McDonnell, it is possible to classify an eye as subclinicalkeratoconus if the simulated central corneal power is greater than 47.2D but less than 48.7 D, with an inferior-superior dioptric asymmetrygreater than 1.4 D, but less than 1.9 D. Eyes presenting central cornealcurvature exceeding 48.7 D, as well as inferior-superior dioptricasymmetry greater than 1.9 D can be classified as “true keratoconus”; 2)The distance visual acuity (DVA) of the subclinical KC eye with value of1.0 (without correction or with spherical and/or cylindricalcompensation<1.50 D).

Exclusion criteria included: 1) Previous surgical intervention in theanterior segment, or childhood corneal trauma or corneal-conjunctivaldisease; 2) Existence of active or systemic inflammation, or oculardisease, or current treatment with systemic or local anti-inflammatorydrugs; 3) Hepatic, renal, hematologic, and immunologic diseases,disorders of thyroid function, uncontrolled diabetes, infections in thedays preceding to the sample collection and solid tumors; as they mayinterfere with the results of the study of molecular markers of innateimmunity.

Patients and Control Subjects in Cohort B

We have designed a prospective, cross sectional study in which 53bilateral KC patients (106 KC eyes), 24 relatives (48 eyes), 13 PMDpatients (26 eyes) and 34 control subjects (68 eyes) were enrolled.Bilateral KC patients (56% males; mean age, 33.3±8.4 years) and PMDpatients (69% males; mean age, 47.7±8.9 years) were selected from adatabase of ectatic corneal patients at Servizo Galego de Saúde,Complexo Hospitalario de Santiago de Compostela, Spain. Relatives (52%males; mean age, 26.7±9.9 years) subjects had family history of KC, butno topographic alterations. Control subjects (41% males; mean age,30.5±9.6 years) had not family history of KC or eye rubbing, and notopographic alterations. We studied both eyes from each KC or PMDpatient, relative or control subject. Patients, relatives and controlsubjects were asked not to wear their contact lenses for a week prior tothe study. All patients and control subjects were expressly cited forthe purposes of this study, and the same researcher performed allexaminations. Data collected included gender, age, patient's ocularhistory, medical history (allergy, eye rubbing), and family history ofKC. This research was carried out in accordance with the Declaration ofHelsinki of the World Medical Association (2008) and approved by theEthics Committee of Research at Servizo Galego de Saúde. Informedconsent was obtained from each patient or control subject after fullexplanation of the procedures.

Inclusion criteria were: 1) Bilateral KC patients (2 KC eyes).Rabinowitz/McDonnell criterion was used for the KC diagnosis (RabinowitzY. S. Keratoconus. Surv Ophthalmol. 1998; 42:297-319). According to thevideokeratographic guidelines proposed by Rabinowitz & McDonnell, it ispossible to classify an eye as subclinical keratoconus if the simulatedcentral corneal power is greater than 47.2 D but less than 48.7 D, withan inferior-superior dioptric asymmetry greater than 1.4 D, but lessthan 1.9 D. Eyes presenting central corneal curvature exceeding 48.7 D,as well as inferior-superior dioptric asymmetry greater than 1.9 D canbe classified as “true keratoconus”; 2) The distance visual acuity (DVA)of the subclinical KC eye with value of 1.0 (without correction or withspherical and/or cylindrical compensation<1.50 D). 2) PMD patients (2 KCeyes). PMD was considered when we identified a marked central cornealflattening along the vertical axis with severe against-the-ruleastigmatism and marked steepening of the inferior peripheral cornea andtypically shows a crab-claw pattern. 3) Relatives: first-degree relativeof patients with ectatic disorders (PMD or KC) but without topographicalterations.

Exclusion criteria included: 1) Previous surgical intervention in theanterior segment, or childhood corneal trauma or corneal-conjunctivaldisease; 2) Existence of active or systemic inflammation, or oculardisease, or current treatment with systemic or local anti-inflammatorydrugs; 3) Hepatic, renal, hematologic, and immunologic diseases,disorders of thyroid function, uncontrolled diabetes, infections in thedays preceding to the sample collection and solid tumors; as they mayinterfere with the results of the study of molecular markers of innateimmunity.

Clinical Variables

-   -   Epidemiological variables: age, gender, KC year of diagnosis,        personal and family history,    -   Therapeutic variables: antihistamines, systemic or local        anti-inflammatory or antibiotic.    -   Variables associated to KC: ocular itching and its intensity,        eye rubbing and its intensity, AVL and AVP with and without        optical correction, biomicroscopical signs, conjunctival        hyperemia scale, Schirmer test, contact lens wear.    -   Topographic variables: PDC (simK), K max, K min, K1, K2, Ka, Dk,        MPD and distance to the center. Classification of Krumeich and        Classification of CLEK for the KC grade. Elevation and        aberrometric values.

Study Groups

For comparative analysis, we have defined the following groups:

Cohort A:

Group 1: controls without topographical alterations.Group 2: subclinical KC.

Group 3: KC. Cohort B:

Group 1: controls without topographical alterations.

Group 2: Bilateral KC. Group 3: PMD.

Group 4: relatives without topographical alterations.

These groups were established in order to determine the predictive valueof TLRs as a biomarker of risk for onset or progression of KC or PMD.

Instrumental

Basic examination instruments were a Topcon biomicroscope, Topconrefraction column, and alphabetic Snellen visual acuity test. Asspecific examination instruments we used a TOPCON CA-100 System (TopconMedical Systems, Inc., NJ, USA), and an Orbscan II corneal topographer(Orbtek, Utah, USA).

Procedure

Our protocol collected the following information: elapsed time fromdiagnosis of KC in the first eye, laterality, itching and rubbing,family history of KC and allergies.

After obtaining the best correction, the biomicroscopy exam wasperformed to detect signs of KC. The corneal topography study wasperformed using the TOPCON and the Orbscan topographers. Fivequantitative topographic parameters were analyzed: simulated keratometry(K) readings, posterior elevation and the thinnest point pachymetry ofthe cornea.

TLR2 and TLR4 Expression Analysis

TLR2 and 4 expression analyses were performed by flow cytometry inconjunctival and corneal cells, withdrawn from all control subjects andsubclinical KC patients by means of a PVA foam surgical spear (SOFTCELL®, OASIS®, CA, USA). For the expression analysis of TLR2 and TLR4,conjunctival and corneal cells were separated by their forward and sidescattering signal characteristics. FITC-TLR2 antibody (IMMUNOSTEP,Salamanca, Spain) and PE-TLR4 antibody (IMMUNOSTEP, Salamanca, Spain)were used to quantify TLR expression. Samples were analyzed on aFACSAria flow cytometer (BD Biosciences, NJ, USA). Cell fluorescence wasmeasured immediately after staining, and data were analyzed with the useof FACSDiva software (BD Biosciences, NJ, USA). For cohort A, meanexpression of TLR2 and TLR4 in conjunctival (1250 events) and cornealcells (500 events) was analyzed and expressed as AFU (arbitraryfluorescence units). For cohort B, mean expression of TLR2 and TLR4 inconjunctival (2000 events) and corneal cells (1000 events) was analyzedand expressed as AFU (arbitrary fluorescence units).

Statistical Analysis

The results were expressed as percentages for categorical variables andas mean (SD) or median [quartiles] for the continuous variablesdepending on their normal distribution or not, respectively. TheKolmogorov-Smirnov test was used for testing the normality of thedistribution. Proportions were compared using the chi-square test, whilethe continuous variables between groups were compared with the Student'st-(variables with normal distribution) or the Mann-Whitney (variableswith non-normal distribution) tests. ANOVA was used to analyze therelationship between study groups and TLR2 and TLR4 in corneal andconjunctival cells. Receiver operating characteristic (ROC) curves wereconfigured to establish cut-off points of TLR2 and TLR4 in corneal andconjunctival cells that optimally predicted risk of appearance orprogression of KC or PMD. A value of p<0.05 was considered to bestatistically significant. The statistical analysis was conducted usingSPSS 16.0 for Mac.

Example 1 Clinical Features for Cohort A

No gender-related statistical differences were detected between thestudy groups, however patients with KC were older and sowed morefrequent history of KC (Table 1). The elapsed time from the firstdiagnosis of KC eye ranged from 1 to 30 years (mean, 8.3±6.2 years).Twenty-three KC patients (46%) and 19 control subjects (51.4%) reportedallergy disease and 15 KC patients (30%) a family history of KC.Thirteen control subjects (35.1%) and 31 patients (62%) reported itchyeyes. For patients who reported itchy eyes, 71% admitted frequent andvigorous eye rubbing on the KC eye. Thirteen control subjects (35.1%)also reported eye rubbing.

Table 1 shows the mean K2 values for all study groups. Mean K2 washigher in the KC eye versus the other study groups (p<0.0001).

Descriptive Study

TABLE 1 Baseline clinical characteristics and TLR conjunctival andcorneal cell expression in study groups of cohort A. Variable ControlSubclinical KC KC p value Age (years) 28.5 ± 4.7 33.3 ± 9.5 33.3 ± 9.50.029 Gender (% males) 45.9 64.0 64.0 0.538 History of KC (%) 0  30.030.0 0.002 Central K (diopters) 43.4 [41.8-45.3] 43.6 [42.3-44.7] 48.1[45.1-52.1] <0.0001 Minimum thickness point (μm) 552.0 [513.5-567.0]519.0 [487.5-546.0] 480.0 [417.0-502.0] <0.0001 Posterior elevation (μm)0.038 [0.024-0.036] 0.036 [0.029-0.047] 0.089 [0.062-0.122] <0.0001 K2(diopters) 43.7 [42.3-45.3] 44.0 [43.0-44.8] 48.0 [46.4-50.5] <0.0001TLR2 conjunctiva (AFU) 213 [90-295] 207 [124-398] 422 [178-1065] <0.0001TLR4 conjunctiva (AFU) 1581 [1281-2265] 1922 [1574-2778] 2377[1817-3032] <0.0001 TLR2 cornea (AFU) 185 [113-286] 977 [647-1330] 2569[2071-3808] <0.0001 TLR4 cornea (AFU) 1654 [1134-2587] 2569 [2071-3808]4125 [3233-5076] <0.0001 Values are expressed in median [quartiles]. KC:Keratoconus; TLR2: Toll-Like Receptor 2); TLR4: Toll-Like Receptor 4);AFU: Arbitrary Fluorescence Unit

TLR2 and TLR4 Expression in Corneal and Conjunctival Cells for Cohort A

Levels of TLR2 and TLR4 expression in KC, subclinical KC and controlgroups in both corneal and conjunctival cells are also shown in table 1.TLR2 and TLR4 expression in both corneal and conjunctival cells washigher in the KC groups. Moreover, the higher the expression of TLR2 andTLR4 was the higher the progression of KC (table 1). However, the maindifference of TLR expression from the control groups to subclinical KCand KC groups was found for the expression of TLR2 and TLR4 in cornealcells. Therefore, predictive analyses were focused exclusively on theexpression of TLR2 and TLR4 in corneal cells.

Predictive Value of TLR2 and TLR4 Expression in Corneal Cells for Riskof Onset and Progress to KC for Cohort A

According to the ROC analysis, TLR2 expression in corneal epithelialcells may predict with high sensitivity and specificity the probabilityof KC (both subclinical KC and KC) compared to the controls (area underthe curve 0.995, 95% CI: 0.987-1.000; p<0.0001). Likewise, TLR2expression in corneal cells is also useful for predicting with highsensitivity and specificity the probability of subclinical KC comparedto the controls (area under the curve 0.989, 95% CI: 0.975-1.000;p<0.0001). Finally, corneal TLR2 expression also predict with the highsensitivity and specificity the probability of no subclinical KCcompared to the KC (area under the curve 0.893, 95% CI: 0.834-0.953;p<0.0001). In summary, table 2 shows the sensitivity and specificity forthe utility of several cut-off points for TLR2 expression in cornealcells for detecting the risk of onset and progression of KC.

TABLE 2 Sensitivity and specificity values for the utility of TLR2expression in corneal cells for detecting the risk of onset ofprogression of KC for cohort A. TLR2 expression (AFU) SENSITIVITYSPECIFICITY Control vs. KC patients (Subclinical KC + KC) <330 AFU 99%84% 330-660 AFU 89-98%    85-99%    >660 AFU 88% 100%  Control vs.Subclinical KC <330 AFU 98% 84% 330-660 AFU 75-97%    85-99%    >660 AFU74% 100%  Subclinical KC vs. KC <330 AFU 100%   2% >660 AFU 100%  26%660-1500 AFU 69-99%    27-87%    >1500 AFU 68% 88%On the other hand, according to the ROC analysis, TLR4 expression incorneal epithelial cells may also predict with high sensitivity andspecificity the probability of KC (both subclinical KC and KC) comparedto the controls (area under the curve 0.846, 95% CI: 0.776-0.915;p<0.0001). Likewise, TLR4 expression in corneal cells is also useful forpredicting with high sensitivity and specificity the probability ofsubclinical KC compared to the controls (area under the curve 0.756, 95%CI: 0.653-0.860; p<0.0001). Finally, corneal TLR4 expression alsopredict with the high sensitivity and specificity the probability of nosubclinical KC compared to the KC (area under the curve 0.767, 95% CI:0.673-0.860; p<0.0001). Table 3 shows the sensitivity and specificityfor the utility of several cut-off points for TLR4 expression in cornealcells for detecting the risk of onset of progression of KC.In conclusion, TLR2 expression in corneal cells showed higher predictivevalue for detecting the risk of onset and progression of KC than theexpression of TLR4.

TABLE 3 Sensitivity and specificity values for the utility of TLR4expression in corneal cells for detecting the risk of onset ofprogression of KC for cohort A. TLR4 expression (AFU) SENSITIVITYSPECIFICITY Control vs. KC patients (Subclinical KC + KC) <1500 AFU 94%46% 1500-4000 AFU 36-93%    45-96%    >4000 AFU 35% 97% Control vs.Subclinical KC <1500 AFU 88% 46% 1500-4000 AFU 23-87%    45-96%    >4000AFU 22% 97% Subclinical KC vs. KC <1500 AFU 100%  22% >4000 AFU 520% 80% >5000 AFU 28% 90%

Correlation Between TLR2 and TLR4 Expression in Corneal Cells andClinical Parameters of KC Progression (K2 and Kc) for Cohort A

The expression levels of TLR2 and TLR4 were analyzed in cornea cellsfrom clinical and subclinical KC patients regarding two quantitativeparameters related to the progression of keratoconus, namely K2 and Kc.A simpler linear regression analysis was made calculating bivariatecorrelations with Pearson test. The results are expressed as correlationcoefficient r:

K2

TLR2; r=0.525; p<0.0001TLR4; r=0.359; p<0.001

Kc

TLR2; r=0.439; p<0.0001TLR4; r=0.379; p<0.0001

These results confirm an association between TLR2 and TLR4 expression incorneal cells with the progression and the severity of subclinical andclinical KC.

Example 2 Clinical Features for Cohort B

No gender-related statistical differences were detected between thestudy groups, however patients with PMD were older and KC patientsshowed more frequent history of ectatic disorders than control subjectsand PMD patients (table 4). Twenty-eight KC patients (65%), 6 PMDpatients (46%), 9 relatives (39%) and 14 control subjects (41.2%)reported allergy disease. Likewise, the 88% of KC patients, 84% of PMDpatients, 39% of relatives and 26% of control subjects reported itchyeyes. For patients who reported itchy eyes, the 76% of KC patients, 76%of PMD patients, 35% of relatives and 35% of control subjects admittedfrequent and vigorous eye rubbing on the KC eye.

Table 4 shows the median K2, central K, minimum thickness point andposterior elevation values for all study groups. Median K2 and central Kwere higher in the KC eyes versus the other study groups (p<0.0001).Likewise, posterior elevation was higher in the KC and PMD eyes versuscontrol and relative subjects (p<0.0001). By contrast, the minimumthickness point was lower for KC and PMD eyes compared to the othergroups (p<0.0001).

Descriptive Study

TABLE 4 Baseline clinical characteristics and TLR conjunctival andcorneal cell expression in study groups of cohort B. DESCRIPTIVE STUDYVariable Control PMD KC Relatives p value Age (years) 30.5 ± 9.6 47.7 ±8.9 33.3 ± 8.4 26.7 ± 9.9 <0.0001 Gender (% males) 41.2 69.2 55.8 52.20.335 History of KC (%) 0   7.7 25.6 100   <0.0001 Central K (diopters)44.0 [42.7-45.3] 43.5 [41.1-45.5] 47.8 [44.2-51.7] 44.3 [43.7-45.0]<0.0001 Minimum thickness point (μm) 560.0 [536.0-582.5] 496.5[410.8-544.0] 471.0 [400.0-509.0] 544.5 [512.5-573.0] <0.0001 Posteriorelevation (μm) 0.029 [0.021-0.038] 0.085 [0.049-0.107] 0.087[0.056-0.117] 0.027 [0.019-0.037] <0.0001 K2 (diopters) 44.4 [43.0-45.8]45.5 [44.1-50.8] 48.6 [45.5-51.7] 44.5 [43.7-45.2] <0.0001 TLR2conjunctiva (AFU) 733 [560-946] 815 [669-1163] 1184 [943-1491] 1178[655-1441] <0.0001 TLR4 conjunctiva (AFU) 2380 [1781-3283] 4080[3392-4610] 4230 [3496-5163] 3331 [2702-4000] <0.0001 TLR2 cornea (AFU)580 [413-976] 743 [639-1106] 1782 [1254-3119] 891 [575-1316] <0.0001TLR4 cornea (AFU) 1572 [949-2353] 2251 [1711-2982] 3812 [2774-5408] 1904[1466-2522] <0.0001 Values are expressed in median [quartiles]. KC:Keratoconus; PMD: Pellucid marginal degeneration; TLR2: Toll-LikeReceptor 2; TLR4: Toll-Like Receptor 4; AFU: Arbitrary FluorescenceUnits.

TLR2 and TLR4 Expression in Corneal and Conjunctival Cells for Cohort B

Levels of TLR2 and TLR4 expression in KC, PMD, relatives and controlgroups in both corneal and conjunctival cells are also shown in table 4.TLR2 and TLR4 expression in both corneal and conjunctival cells washigher in the KC group respect to the other groups. Moreover, PMD groupalso showed higher expression of TLR2 in corneal cells and TLR4 in bothcorneal and especially conjunctival cells than the control group (table4). Interestingly, relatives showed higher expression of TLR2 and TLR4in both corneal and conjunctival cells than control subjects, but thisexpression is much lower than in patients KC (table 4). However, themain difference of TLR expression from the control group to KC group wasfound for the expression of TLR2 and TLR4 in corneal cells. On the otherhand, the main difference of TLR expression from the control group toPMD group was found for the expression of TLR4 in conjunctival cells.Therefore, predictive analyses were focused exclusively on theexpression of TLR2 and TLR4 in corneal cells for KC, and TLR4 inconjunctival cells for PMD.

Predictive Value of TLR2 and TLR4 Expression in Corneal Cells for Riskof Onset and Progress to KC for Cohort B

According to the ROC analysis, TLR2 expression in corneal epithelialcells may predict with high sensitivity and specificity the probabilityof KC compared to the controls (area under the curve 0.874, 95% CI:0.815-1.000; p<0.0001). Likewise, TLR2 expression in corneal cells isalso useful for predicting with high sensitivity and specificity theprobability of KC compared to relatives (area under the curve 0.802, 95%CI: 0.723-0.882; p<0.0001). In summary, table 5 shows the sensitivityand specificity for the utility of several cut-off points for TLR2expression in corneal cells for detecting the risk of onset andprogression of KC.

On the other hand, according to the ROC analysis, TLR4 expression incorneal epithelial cells may also predict with high sensitivity andspecificity the probability of KC compared to the controls (area underthe curve 0.877, 95% CI: 0.818-0.936; p<0.0001). Likewise, TLR4expression in corneal cells is also useful for predicting with highsensitivity and specificity the probability of KC compared to relatives(area under the curve 0.882, 95% CI: 0.821-0.943; p<0.0001).

TABLE 5 Sensitivity and specificity values for the utility of TLR2expression in corneal cells for detecting the risk of onset orprogression of KC in cohort B. TLR2 expression (AFU) SENSITIVITYSPECIFICITY Control subjects vs. KC patients <327 AFU 99% 86% 327-1269AFU 75-98%    76-91%    >1269 AFU 89% 92% Relatives vs. KC patients <521AFU 95% 84% 521-1352 AFU 71-94%    28-88%    >1352 AFU 71% 91%

Table 6 shows the sensitivity and specificity for the utility of severalcut-off points for TLR4 expression in corneal cells for detecting therisk of onset of progression of KC.

In conclusion, TLR2 and TLR4 expression in corneal cells showed a highpredictive value for detecting the risk of onset and progression of KC.

TABLE 6 Sensitivity and specificity values for the utility of TLR4expression in corneal cells for detecting the risk of onset orprogression of KC in cohort B. TLR4 expression (AFU) SENSITIVITYSPECIFICITY Control subjects vs. KC patients <1508 AFU 95% 53% 1508-4000AFU 42-94%    52-96%    >4000 AFU 41% 97% Relatives vs. KC patients<1697 AFU 95% 41% 1697-3794 AFU 51-99%    42-99%    >3794 AFU 50% 100% 

Predictive Value of TLR4 Expression in Conjunctival Cells for Risk ofOnset and Progress to PMD for Cohort B

Finally, according to the ROC analysis, TLR4 expression in conjunctivalepithelial cells may also predict with high sensitivity and specificitythe probability of PMD compared to the control subjects (area under thecurve 0.823, 95% CI: 0.733-0.913; p<0.0001). Table 7 shows thesensitivity and specificity for the utility of several cut-off pointsfor TLR4 expression in conjunctival cells for detecting the risk ofonset of progression of KC.

In conclusion, TLR4 expression in conjunctival cells showed also highpredictive value for detecting the risk of onset and progression of PMD.

TABLE 7 Sensitivity and specificity values for the utility of TLR4expression in conjunctival cells for detecting the risk of onset orprogression of PMD in cohort B. Control subjects vs. PMD patients TLR4expression (AFU) SENSITIVITY SPECIFICITY <1543 AFU 100% 82% 1543-4500AFU 31-99%  52-92%    >4500 AFU  31% 93%

Correlation Between TLR2 and TLR4 Expression in Corneal Cells andClinical Parameters of KC Progression (K2, Kc and Kmax) for Cohort B

We analyzed the expression levels of TLR2 and TLR4 in corneal cells fromKC patients regarding three quantitative parameters related to theprogression of keratoconus, namely K2, Kc and Kmax and made a simplerlinear regression analysis calculating bivariate correlations withPearson test. The results are expressed as correlation coefficient (r):

K2

TLR2; r=0.286; p<0.0001TLR4; r=0.249; p<0.0001

Kc

TLR2; r=0.266; p<0.0001TLR4; r=0.286; p<0.0001

Kmax

TLR2; r=0.403; p<0.0001TLR4; r=0.263; p<0.0001These results confirm an association between TLR2 and TLR4 expression incorneal cells with the progression and the severity of KC.

Correlation Between TLR4 Expression in Conjunctival Cells and ClinicalParameters of PMD Progression (Kmax) for Cohort B

On the other hand, we also analyzed the expression levels of TLR4 inconjunctival cells from PMD patients regarding Kmax, a parameter relatedto the progression of PMD. We made a simpler linear regression analysiscalculating bivariate correlations with Pearson test. The results areexpressed as correlation coefficient (r):

Kmax

TLR4; r=0.327; p<0.0001

In summary, these results also confirm an association between TLR4expression in conjunctival cells with the progression and the severityof PMD.

1-31. (canceled)
 32. An in vitro method for diagnosing an ectaticdisease of the cornea in a subject, or for determining the risk ofdeveloping an ectatic disease of the cornea in a subject, or fordetermining the clinical outcome of a subject suffering from an ectaticdisease of the cornea or for selecting a subject to be treated with atherapy for an ectatic disease of the cornea which comprises: a)determining the expression level of TLR2 and/or TLR4 in a sample fromsaid subject; and b) comparing said expression level with a referencevalue wherein, if the expression level of TLR2 and/or TLR4 is higherthan said reference value is indicative that the subject suffers from anectatic disease of the cornea or that the subject has high risk ofdeveloping an ectatic disease of the cornea or that the subject has anegative clinical outcome or that the subject is candidate to be treatedwith a therapy for an ectatic disease of the cornea.
 33. The in vitromethod according to claim 32 wherein said ectatic disease of the corneais selected from subclinical keratoconus, clinical keratoconus, pellucidmarginal degeneration, keratoglobus and ectasia post refractive cornealsurgery.
 34. The in vitro method according to claim 33, wherein saidectatic disease of the cornea is subclinical keratoconus, in which casethe reference value corresponds to the expression levels of TLR2 and/orTLR4 measured in a sample from a healthy subject.
 35. The in vitromethod according to claim 33, wherein said ectatic disease of the corneais clinical keratoconus, in which case the reference value correspondsto the expression levels of TLR2 and/or TLR4 measured in a sample from asubject suffering from subclinical keratoconus.
 36. The in vitro methodaccording to claim 33, wherein said ectatic disease of the cornea ispellucid marginal degeneration, in which case the reference valuecorresponds to the expression levels of TLR2 and/or TLR4 measured in asample from a healthy subject.
 37. The in vitro method according toclaim 32, wherein the sample from said subject is selected fromconjunctival and corneal tissue.
 38. The in vitro method according toclaim 32, wherein said expression level comprises determining the levelof mRNA encoded from the TLR2 and/or TLR4 gene or determining the levelof the TLR2 and/or TLR4 protein.
 39. The in vitro method according toclaim 38, wherein the protein level is determined by a method selectedfrom immunohistochemistry, Western blot, flow cytometry or by ELISA. 40.The in vitro method for diagnosing an ectatic disease of the cornea in asubject according to claim 32 which also comprises determining at leastone parameter selected from: diopters, corneal thickness and cornealelevation.
 41. The in vitro method for determining the risk ofdeveloping an ectatic disease of the cornea in a subject according toclaim 32, wherein the subject has been previously diagnosed as having arefractive defect.
 42. The in vitro method for determining the risk ofdeveloping an ectatic disease of the cornea in a subject according toclaim 32, wherein said subject has developed a previous ocular pathologyselected from: ocular itching, eye rubbing, biomicroscopic signs andconjunctival hyperemia.
 43. The in vitro method for selecting a subjectto be treated with a therapy for an ectatic disease of the corneaaccording to claim 32, wherein said therapy is selected from cornealcrosslinking, intracorneal rings and refractive surgery.
 44. An in vitromethod for determining whether a subject suffering from a refractivedefect is a candidate for a refractive surgery therapy or fordetermining the risk that a subject suffers ectasia following refractivesurgery which comprises: a) determining the expression level of TLR2and/or TLR4 in a sample from said subject; and b) comparing saidexpression level with a reference value wherein if the expression levelof TLR2 and/or TLR4 is higher than said reference value is indicativethat said subject is not candidate to be treated with a refractivesurgery for said refractive defect or is indicative that said subjectshows high risk of suffering ectasia following refractive surgery.
 45. Acomposition or kit for diagnosis of an ectatic disease of the corneawhich comprises an antibody, a polypeptide, a primer and/or a probewhich specifically bonds to TLR2 and/or TLR4.
 46. A method fordetermining the diagnosis of an ectatic disease of the cornea in asubject, for determining the risk of developing an ectatic disease ofthe cornea in a subject, for determining the clinical outcome of asubject suffering from an ectatic disease of the cornea or for selectinga subject to be treated with a therapy for an ectatic disease of thecornea comprising the use of the composition or kit according to claim45.
 47. The in vitro method according to claim 44, wherein the samplefrom said subject is selected from conjunctival and corneal tissue. 48.The in vitro method according to claim 44, wherein said expression levelcomprises determining the level of mRNA encoded from the TLR2 and/orTLR4 gene or determining the level of the TLR2 and/or TLR4 protein. 49.The in vitro method according to claim 48 wherein the protein level isdetermined by a method selected from immunohistochemistry, Western blot,flow cytometry or by ELISA.